Nanochannel arrays and their preparation and use for high throughput macromolecular analysis

ABSTRACT

Nanochannel arrays that enable high-throughput macromolecular analysis are disclosed. Also disclosed are methods of preparing nanochannel arrays and nanofluidic chips. Methods of analyzing macromolecules, such as entire strands of genomic DNA, are also disclosed, as well as systems for carrying out these methods.

RELATED APPLICATIONS

[0001] This patent application claims the benefit of priority to U.S.Provisional Patent Application No. 60/307,666, filed on Jul. 25, 2001.DARPA Grant Number MDA972-00-1-0031 supported work that led to portionsof the inventions described herein. Accordingly, the U.S. Government mayhave rights in these inventions.

BACKGROUND

[0002] The present invention relates to a nanochannel array. The presentinvention also relates to a method of preparing nanochannel arrays. Thepresent invention also relates to nanofluidic chips containingnanochannel arrays. The present invention also relates to a systemsuitable for high throughput analysis of macromolecules. The presentinvention also relates to a method of analyzing at least onemacromolecule by using a nanochannel array.

[0003] In the newly emerging field of bionanotechnology, extremely smallnanofluidic structures, such as channels, need to be fabricated and usedas arrays for the manipulation and analysis of biomolecules such as DNAand proteins at single molecule resolution. In principle, the size ofthe cross sectional area of channels should be on the order of the crosssectional area of elongated biomolecules, i.e., on the order of 1 to 100square nanometers, to provide elongated (e.g., linear, unfolded)biomolecules that can be individually isolated, yet analyzedsimultaneously by the hundreds, thousands, or even millions. Likewise,it is also desirable that the length of the channels should be longenough to accommodate the longest of macromolecules, such as an entirechromosome, which can be on the order of 10 centimeters long (e.g.,chromosome 1 of the human genome having 250 million base pairs). Thepresent inventors and others have recently been concerned about suchproblems and their possible solutions, as reported in: O. Bakajin, etal., Anal. Chem. 73 (24), 6053 (2001), J. O. Tegenfeldt, et al., Phys.Rev. Lett. 86 (7), 1378 (2001), J. Han et al., Science 288, 1026 (2000),and S. R. Quake et al., Science 290 (5496), 1536 (2000).

[0004] It is important to efficiently and reliably construct arrays ofmany thousands, or even millions of channels in an array for thesimultaneous isolation and analysis of up to thousands or millions ofindividual macromolecules. Such large arrays of isolated macromoleculescould, in principle, be analyzed with presently available twodimensional area detectors, such as charge-coupled devices (CCDs).Together with automated data-processing collection and image analysissoftware, the simultaneous characterization of up to thousands ormillions of macromolecules would be an extremely powerful tool formacromolecular analysis, such as population distribution analysis ofmacromolecular size, chemical composition, and DNA sequencing.

[0005] Because individual macromolecules could in principle be isolatedand analyzed in a single channel, heterogeneity of a sample containing amultitude of macromolecules can be readily discerned. This would beparticularly useful for identifying single nucleotide polymorphisms(SNP) on a single chromosome. In contrast, traditional population basedassays require time-consuming DNA amplification methods to preparemultiple copies of a nucleic acid macromolecule to carry out SNPanalysis. If available, a chromosomal analysis system incorporatingnanochannel arrays could perform SNP analysis much more quickly than anymethod presently available.

[0006] Nanochannel arrays having the proper dimensions for carrying outthe simultaneous isolation and analysis of a multitude of elongatedmacromolecules have been heretofore unavailable. Accordingly, there isan urgent need to provide nanochannel arrays having at least three keydimensional qualities: (1) the channels should have a sufficiently smalldimension to elongate and isolate macromolecules; (2) the channelsshould have a sufficiently long dimension to permit the instantaneousobservation of the entire elongated macromolecule; and (3) a high numberof channels should be provided to permit the simultaneous observation ofa high number of macromolecules. In addition, it would be desirable forthe elongated and isolated macromolecules to remain indefinitely in sucha state at ambient conditions even after the field which is used totransport the macromolecules into the channels (e.g., electric field) isturned off. This feature would permit the macromolecules to be analyzedwith techniques that require times longer than the residence time of themacromolecule under the influence of the field. This feature would alsopermit the analysis of macromolecules without having to subject them toa field.

[0007] Methods for analyzing macromolecules (e.g., polymers) have beenpreviously disclosed, however none uses a nanochannel array having thethree key dimensional qualities as described supra. U.S. Pat. No.5,867,266 discloses a micro optical system having a plurality ofcoplanar micron-to-millimeter scale sample channels prepared usingphotolithography and an artificial gel material comprising amultiplicity of pillar structures in each micron-to-millimeter widesample channel. The large channel width makes this system unsuitable asa nanochannel array.

[0008] Likewise, methods for analyzing macromolecules (e.g., polymers)by isolating them in channels more narrow than this disclosed in U.S.Pat. No. 5,867,266, however none uses a nanochannel array having thethree key dimensional qualities as described supra. In WO 00/09757,several of the inventors of the present patent application disclose asystem for optically characterizing single polymers that are transportedin a straightened form through a channel. In U.S. Pat. No. 6,355,420, asystem is disclosed for analyzing polymers that are transported in astraightened form through a plurality of (at least 50) channels. Whileboth of these disclosures are directed towards analysis of singlemacromolecules aligned in one or more channels, neither of thesedocuments discloses the simultaneous observation of a high number ofmacromolecules in a multitude of channels.

[0009] Thus, there remains the problem of providing suitable nanochannelarrays that are useful in a variety of macromolecular analysis. Methodsfor analyzing macromolecules (e.g., polymers) by isolating them in anarrow channel have been previously disclosed, however none uses ananochannel array having the three key dimensional qualities asdescribed supra, primarily because, until now, fabrication techniquesfor constructing such a nanochannel array were not available.

[0010] In creating ultra-small nanofluidic structures, e.g. for singlebiomolecule analysis, at least two problems need to be solved: reductionof size and creation of sealed fluidic channels. As reported by one ofthe present inventors, NIL is a parallel high-throughput technique thatmakes it possible to create nanometer-scale features over largesubstrate surface areas at low cost. (S. Y. Chou et al., Appl. Phys.Lett. 67 (21), 3114 (1995) and S. Y. Chou et al., Science 272, 85(1996)) Current sealing techniques such as wafer bonding (M. Stjernstromet al., J. Micromech. and Microeng. 8 (1), 33 (1998)), and softelastomer sealing (H. P. Chou et al., Proc. Nat. Acad. Sci. USA 96 (1),11 (1999), are suitable for relatively large planar surfaces and providean effective seal. Wafer bonding requires an absolutely defect free andflat surface, and elastomer sealing suffers from clogging due to softmaterial intrusion into the channels. Within extremely small confiningstructures, biological samples are also much more sensitive to issuessuch as hydrophobicity and the homogeneity of the material constructingthe fluidic structure.

[0011] Recently developed techniques using “place-holding” sacrificialmaterials such as polysilicon (S. W. Turner et al., J. Vac. Sci. andTechnol. B 16(6), 3835 (1998)) and polynorbornene (D. Bhusari et al., J.Microelectromech. Syst. 10 (3), 400 (2001)) have gained popularity tocreate sealed small hollow fluidic structures. However, steps needed inremoving the sacrificial materials such as heating the substrate up to200-400° C. or wet etching limits the use of certain materials anddownstream fabrication processes.

[0012] As provided herein, the present invention achieves the goal ofproviding nanochannel arrays suitable for performing high throughputmacromolecular analysis. Interferometric lithography (IL), nanoimprintlithography (NIL), and non-isotropic deposition techniques are used toprepare nanochannel arrays having hundreds of thousands to more than amillion enclosed channels having the desired key dimensions across thesurface of a silicon wafer substrate.

[0013] In one aspect of the present invention, there are providednanochannel arrays including a surface having a plurality of channels inthe material of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least some of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed.

[0014] In a further aspect of the present invention, methods ofpreparing nanochannel arrays are disclosed, which include the steps of:providing a substrate having a surface; forming a plurality of channelsin the material of the surface; and depositing a sealing material on theplurality of channels to surmount the plurality of channels to rendersuch channels at least substantially enclosed, the substantiallyenclosed channels having a trench width of less than 150 nanometers anda trench depth of less than 200 nanometers.

[0015] In another aspect of the invention, there are providednanofluidic chips including: a) nanochannel array, including: asubstrate having a surface; a plurality of parallel channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least some of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast some of the channels are capable of admitting a fluid; b) at leastone sample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid; and c) atleast one waste reservoir in fluid communication with at least one ofthe channels, said waste reservoir capable of receiving a fluid.

[0016] In yet another embodiment of this invention, there are providedsystems for carrying out analysis. In exemplary embodiments, theseinclude: A) a nanofluidic chip, including: a) nanochannel array,including: a substrate having a surface, a plurality of parallelchannels in the material of the surface, said channels having a trenchwidth of less than about 150 nanometers and a trench depth of less than200 nanometers; at least one of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast one of the channels capable of admitting a fluid; and b) at leastone sample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid; and B) adata processor.

[0017] In another embodiment, methods of analyzing at least onemacromolecule are described which, for example, include the steps of:providing a nanofluidic chip, including: a) nanochannel array,including: a surface having a plurality of parallel channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least one of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast one of the channels capable of admitting a fluid; b) at least onesample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid containingat least one macromolecule; providing the at least one sample reservoirwith at least one fluid, said fluid comprising at least onemacromolecule; transporting the at least one macromolecule into the atleast one channel to elongate said at least one macromolecule; detectingat least one signal transmitted from the at least one elongatedmacromolecule; and correlating the detected signal to at least oneproperty of the at least one macromolecule.

[0018] Cartridges including a nanofluidic chip in accordance with thisinvention are also disclosed herein. Such cartridges are capable ofbeing inserted into, used with and removed from a system such as thoseshown herein. Cartridges useful with analytical systems other than thesystems of the present invention are also comprehended by thisinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 illustrates a cross-section of a nanochannel array havingsubstantially enclosed channels.

[0020]FIG. 2 illustrates a cross-section of a nanochannel array havingcompletely enclosed channels and having sealing material deposited inthe channels.

[0021]FIG. 3 is a scanning electron micrograph of a nanochannel arrayhaving parallel linear channels and open channel ends.

[0022]FIG. 4 illustrates a schematic of a process for depositing sealingmaterial into the channels.

[0023]FIG. 5 illustrates a nanofluidic chip.

[0024]FIG. 6a is a scanning electron micrograph of the substrate used inExample 4 prior to sealing with silicon dioxide.

[0025]FIG. 6b is a scanning electron micrograph of the nanochannel arrayof Example 4 obtained after sealing the substrate with silicon dioxide.

[0026]FIG. 6c is a scanning electron micrograph (top view) of thenanochannel array of Example 5.

[0027]FIG. 6d is a scanning electron micrograph (top view) of thenanochannel array of Example 4.

[0028]FIG. 7a is a scanning electron micrograph of the substrate used inExample 1 prior to sealing with silicon dioxide.

[0029]FIG. 7b is a scanning electron micrograph of the nanochannel arrayof Example 1 obtained after sealing the nanochannel array in 7 a withsilicon dioxide.

[0030]FIG. 7c is a scanning electron micrograph of the substrate used inExample 2 prior to sealing with silicon dioxide.

[0031]FIG. 7d is a scanning electron micrograph of the nanochannel arrayof Example 2 obtained after sealing the nanochannel array of 7 c withsilicon dioxide.

[0032]FIG. 7e is a scanning electronmicrograph of the substrate used inExample 3 prior to sputtering with silicon dioxide.

[0033]FIG. 7f is a scanning electron micrograph of the nanochannel arrayof Example 3 obtained after sputtering the nanochannel array in 7 e withsilicon dioxide.

[0034]FIG. 8a illustrates a sealed channel having a nanoslit in anopaque layer across the bottom of a channel.

[0035]FIG. 8b illustrates a sealed channel having a nanoslit in anopaque layer across the sealing layer, which is oriented perpendicularto the long axis of a nanochannel.

[0036]FIG. 9a shows scanning electron micrographs of the substrate (leftand bottom) and a of the sealed nanochannel array (right) used inExample 14.

[0037]FIG. 9b is the image obtained from the CCD of the 48.5 kb lambdaphage genome (shorter) and 168 kb T4 phage genome (longer) of Example14. Inset: Plot of genome size versus macromolecular contour length.

[0038]FIG. 9c shows a nanochannel array simultaneously elongating,separating, and displaying a plurality of DNA macromolecules ranging insize from 10 kb to 196 kb.

[0039]FIG. 10 illustrates a system for analyzing macromolecules using ananofluidic chip.

[0040] One aspect of the present invention encompasses a nanochannelarray having a plurality of channels that are substantially enclosed. Asshown in FIG. 1, the nanochannel array 100 has a surface 102 thatcontains a plurality of channels 104 in the material of the surface 106.The channels 104 have a wall 110, and a channel center 112. The distancebetween the wall surfaces 110 inside a channel 104 that areperpendicularly opposite to the channel center 112 is defined as thetrench width. The channels 104 are surmounted by a sealing material 108that renders the channels 104 at least substantially enclosed.

[0041] In one embodiment, the channels 104 will not be completelyenclosed and will typically have no sealing material 108 directly abovethe channel center 112, providing an opening in the sealing material tothe channel. The opening may have a variety of shapes. The size of theopening is defined as the minimum distance spanning the opening abovethe channel center 112. In such embodiments, the size of the opening isless than the trench width, and is typically less than ½ of the trenchwidth, more typically less than ⅓ of the trench width, and mosttypically less than ¼ of the trench width. In other embodiments thechannels can be completely enclosed, having sealing material completelycovering the top of the channel and having no opening in the sealingmaterial. In certain embodiments of the present invention, sealingmaterial 108 can extend over the walls 110 and the bottom of thechannels 104, as shown in FIG. 2. In such embodiments, the trench widthis defined as the distance from the surfaces formed by the sealingmaterial adjacent to the walls 114.

[0042] In the present invention, the trench width is typically less than150 nanometers, more typically less than 100 nanometers, and even moretypically less than: 75, 50, 25, and 15 nanometers. In certainembodiments the trench width is about 10 nanometers. In the presentinvention, the trench width is at least 2 nm, and typically at least 5nm.

[0043] In the present invention the channels are at least substantiallyenclosed. “At least substantially enclosed” means that the channels arecompletely enclosed or have an opening in the sealing material that issmaller than ½ the trench width, or have both completely enclosedchannels and openings.

[0044] Channels that are completely enclosed have a trench depth that isdefined as the distance between the surface of the solid material at thebottom of the channel below the channel center 112 to the sealingmaterial above the channel center 112. Embodiments in which the channelshaving an opening have a trench depth defined as the distance from thesurface of the solid material at the bottom of the channel below thechannel center to the position of the opening where the opening size ismeasured. If the opening has more than one position where a minimumdistance can be measured then the position of the opening is the onethat is closest to the bottom of the channel 104.

[0045] In the present invention, the trench depth is less than 200 nm.In certain embodiments, the trench depth is typically less than 175 nm,and more typically less than 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, and25 nm. In certain embodiments the trench depth is about 15 nm. Incertain embodiments the trench depth is at least 2 nm, typically atleast 5 nm, and more typically at least 10 nm.

[0046] In the present invention, the nanochannel arrays can be formed ina substrate, such as a silicon wafer substrate, using a variety offabrication methods as described below. In one embodiment, thenanochannel array has a plurality of parallel linear channels across thesurface of substrate as illustrated by the scanning electron micrographin FIG. 3.

[0047] In certain embodiments, the nanochannel arrays have at least oneend of at least one of the channels can be in fluid communication withat least one reservoir. In these embodiments, at least one channel isconnected directly with at least one reservoir. Alternatively, at leastone channel can be connected with at least one reservoir via aninterconnecting straight or curved microchannel (a channel having awidth, height, or both larger than about a micron), or a channel isconnected with at least one reservoir via an interconnecting nanopillaror micropillar array.

[0048] In certain embodiments, at least two ends of some of the channelsare in fluid communication with at least one reservoir common to thechannels. In these embodiments, at least two ends of some of thechannels can be adjacent or not adjacent. These channels can beconnected directly with at least one reservoir.

[0049] In certain embodiments, at least two channels can be connectedwith at least one reservoir via a common interconnecting straight orcurved microchannel. Alternatively, at least two channels can beconnected with at least one reservoir via a common interconnectingnanopillar or micropillar array.

[0050] In certain embodiments of the present invention, the nanochannelarray has a plurality of channels that are in fluid communication withat least one sample reservoir common to at least some of the channels.By “a plurality of channels” is meant more than two channels, typicallymore than 5, and even typically more than 10, 100, 1000, 10,000 and100,000 channels. In certain embodiments, one sample reservoir can beprovided for all of the channels in the nanochannel array, thus theplurality of channels can be as large as the number of channels that areon the substrate. In a certain embodiments, 100 mm diameter substratescan have about 500,000 parallel linear channels having a periodicity of200 nm, the periodicity being defined as the distance between the middleof two adjacent channels.

[0051] In certain embodiments, the plurality of channels can beconnected directly with at least one reservoir. The connections can be acommon interconnecting straight or curved microchannel. In otherembodiments, a plurality of channels can be connected with at least onereservoir via a common interconnecting nanopillar or micropillar array.

[0052] In certain embodiments of the present invention, thenanonanochannel array contains a plurality of channels that are in fluidcommunication with at least one waste reservoir. Although the pluralityof channels is typically connected directly with at least one wastereservoir, more than one waste reservoir can also be provided. It shouldbe appreciated that the waste reservoir can be used as a samplecollection reservoir. Accordingly, multiple sample collection reservoirscan also be provided on nanochannel arrays. In these embodiments, aplurality of channels can be connected with at least one waste reservoirvia a common interconnecting straight or curved microchannel asdescribed earlier. Likewise, a plurality of channels can be connectedwith at least one waste reservoir via a common interconnectingnanopillar or micropillar array.

[0053] In certain embodiments of the present invention, the nanochannelarray has a plurality of channels that are substantially parallel, inwhich the plurality of channels are substantially in the plane of thesurface of the substrate.

[0054] In certain embodiments of the present invention, the nanochannelarray can contain linear channels. Linear channels are adjacent channelsthat are substantially not interconnected.

[0055] In certain embodiments, the ends of the channels are capable ofadmitting a macromolecule in a fluid. By being capable of admittingmacromolecule means that the channels have at least one opening largeenough to permit the passage of a macromolecule. While a variety ofopenings are envisages, typically such openings can be located at theends of the channels or on the surface of the sealing material throughopenings in the sealing material. Openings in the sealing material canbe provided by subsequent modification of the nanochannel arrays asprovided below.

[0056] In certain embodiments of the present invention, the nanochannelarray contains channels that are capable of transporting a macromoleculeacross their length. The nanochannel arrays can be fitted with a varietyof components to affect macromolecular transport, examples of whichinclude pressure or vacuum gradient drop across the channels,electroosmosis, and electrokinesis.

[0057] While not being bound to a particular theory, it is believed thatmacromolecules typically have a non-linear, three-dimensionalconformation in space (e.g., linear polymers have a random coilconformation in their natural state). Accordingly, it isthermodynamically unfavorable for macromolecules to spontaneouslyelongate and enter channels directly from the environment due to thelarge free energy needed to reduce entropy. For example, a 169 kilobaseT4 phage double stranded genomic DNA chains will form a Gaussian coil ofradius of gyration Rg=(Lp/6)^(1/2)=700 nm in free solution, where L isits calculated contour length and p is the persistence length of about50 nm.

[0058] In certain embodiments, the nanochannel array can containchannels capable of transporting at least one macromolecule across thelength of channels, in which the macromolecule is in an elongated form.Such channels can have openings large enough to permit the entrance ofthe ends of the macromolecules. In certain embodiments, it is preferredthat such channels also have trench widths and trench depths narrowenough to restrict the movement of the macromolecules to primarily onedirection along the surface of the substrate. Preferably such channelsare not interconnected.

[0059] In certain preferred embodiments of the present invention, thenanochannel array is capable of transporting at least one biopolymeracross the length of said channels. In these embodiments, the geometryof the channels permits the biopolymers to enter and move along thechannels in at least one direction. Preferably, the channel surfaces aretreated with a non-sticking agent, as described later, for preventingthe adhesion of macromolecules, such as biopolymers, to the inside ofthe channels.

[0060] In other embodiments, it is preferred that the nanochannel arraycontains channels capable of transporting at least one unfoldedbiopolymer across the length of said channels. While not being bound bya particular theory, when the dimensions of the channels are apparentlylarger than the spatial conformation of the macromolecules, there is atleast a partial amount of elongation of the macromolecules in thechannels. When the dimensions of the channels are at the same order orbelow the persistence length of macromolecules, such as 50 nm for DNAthe macromolecules can be sufficiently elongated in an unfolded fashioninside the channels. When the dimensions of the channels fall in betweenthe above-mentioned two scenarios, macromolecules can be partiallyelongated in these channels. In this case, the macromolecules can befolded, tangled, or both folded and tangled. While it is envisaged thatany macromolecule can be transported in an unfolded fashion in thechannels of the nanochannel array of the present invention, a variety ofsuitable unfolded macromolecules include RNA, DNA, denatured unfoldedpeptide chains, self-assembled chemical polymers, and co-polymer chains,and other biopolymers, and combinations thereof.

[0061] In one preferred embodiment, the channel structures of thenanochannel arrays can be formed from linearly adjacent channel wallsthat span the substrate surface. In other embodiments, the channels canbe formed from pillar structures, self-assembled polymer structures,stacked membrane layers, and nanobeads (particles inside the channels).

[0062] The surface material of the nanochannel arrays can be formed fromalmost any substrate material, such as a conductive material, asemiconductor material, or a non-conductive material. Examples ofconductive materials include metals such as aluminum, gold, silver, andchromium. Examples of semiconductive materials include doped silicondioxide and gallium arsenide. Examples of non-conductive materialsinclude fused silica, silicon dioxide, silicon nitride, glass, ceramics,and synthetic polymers. The foregoing is exemplary only.

[0063] In the present invention, the surface of the nanochannel array istypically the surface of the substrate, such as the surface of a siliconwafer. Alternatively, the surface can be a film, such as one adjacentlysupported by a second substrate. Coating a material on to a secondsubstrate can form films. A suitable coating process includes vapordeposition of a material onto a wafer.

[0064] In certain embodiments of the present invention, the nanochannelarray includes at least one optically opaque material layer adjacent tothe sealing material. The optically opaque material can be situatedbetween the surface material and the sealing layer, it can be situatedinside the channels, it can be situated on top of the sealing material,or a combination of these. While almost any opaque material that can bedeposited as a layer is possible in this embodiment, Aluminum ispreferred. For certain embodiments, it is desirable that the opaquelayer thicknesses are less than about 50 nm thick. For embodimentscontaining nanoslits useful for carrying out near-field imaging of thecontents of the channels, it is desirable to prepare slits smaller than50 nm that are etched through the deposited opaque layer but not throughthe transparent sealing material for maintaining the integrity of theadjacent (underneath) channel. Without being bound by a particulartheory, the optically opaque layer functions as a blocking mask forhigh-resolution near-field excitation. Without being bound to aparticular theory, aluminum is particularly preferred as an opaque layerbecause it has the highest known skin depth of any material at the givenwavelength of the excitation light source rendering the smallestthickness of the blocking layer, hence the shortest distance between theslits and the possible target molecules in channels.

[0065] In certain embodiments of the present invention, the nanochannelarray has at least one near field slit feature above at least onechannel. Such slits should be fabricated as close (less than about 30nm) to the channel as possible without compromising the integrity of theadjacent sealed channels. The thin wall of sealing material between slitopening and channels could be created by FIB milling or controlledmaterial deposition.

[0066] In a further preferred embodiment, the nanochannel array containssealing material adjacent to the channel bottom. Sealing material can beprovided in channel bottom by depositing a suitable sealing materialinto the channels prior to or simultaneously with enclosing thechannels.

[0067] The nanochannel arrays also preferentially have sealing materialadjacent to the channel wall material. In this embodiment, the sealingmaterial can reduce the trench width. This is particularly advantageousfor preparing nanochannel arrays from a variety of substrate surfacesthat contain channels wider than 150 nm in trench width and deeper than200 nm in trench depth. In this embodiment, and as described below,sealing material can be deposited into channels by a variety of methods.One suitable method is E-beam evaporation, which creates a point sourceof material. In E-beam evaporation, a substrate is typically far awayfrom the source compared to the size of the sample, and the angulardistribution of the depositing material is very narrow. To achieve anon-uniform deposition the substrate is tilted at a specific angle. Thechannel walls partially block the deposition of sealing material (like ashadow), and most of the material is be deposited on the channel wallsnear the upper portion of the channel wall. Beyond a critical depth nodeposition will occur as long as the substrate is tilted.

[0068] An alternative and preferred method to provide sealing materialin the channels is sputter deposition. In sputter deposition, thesealing material is deposited at all angles, so the instant growth rateat any point on the surface depends on the percentage of the totaltarget area within its line of sight as is outlined in FIG. 4. Withoutbeing bound to a particular theory, sealing material from a large targetsource 130 that is in close proximity to the substrate surface, cantravel along a variety of trajectories (122, 124, 126, 127) and bedeposited at different positions in the channels. Sputtering istypically used because of the divergent nature of the material beam,thus resulting in the faster deposition of target material at the toppart of the channels instead of at the bottom of the channels (i.e.,surmounting the channel). In time, the sealing material near the top ofthe channels eventually completely encloses the channel, which preventsfurther deposition of sealing material into the channel. In oneembodiment, the resultant sealing material in the channel results in aprofile 128. Suitable sputtering systems are known in the art. Aparticularly suitable sputtering system has a 200 mm SiO2 target sourcewhich provides high surface coverage and uniformity across 100 mmsubstrate.

[0069] The lengths of the channels of the nanochannel array can have awide range. The lengths of the channels can also be the same ordifferent in the nanochannel array. For carrying out macromolecularanalysis using the nanochannel arrays as provided below, it is desirablethat he channels are at least 1 millimeter (mm) longer. More typically,the length of the channels is greater than 1 centimeter (cm), and evengreater than 5 cm, 15 cm, and 25 cm.

[0070] In another aspect of the present invention there is provided amethod of preparing a nanochannel array, which includes the steps offorming a plurality of channels in the material of the surface of asubstrate, and depositing a sealing material to surmount the pluralityof channels to provide at least substantially enclosed channels.Substrates containing a plurality of channels preferably have aperiodicity of 200 nanometers or less, which can be provided byinterferometric lithography and nanoimprint lithography techniques,which are disclosed in U.S. Pat. No. 5,772,905, the complete disclosureof which is incorporated by reference herein. As described earlier,various types of materials can be used to prepare surfaces having aplurality of channels. Suitable substrates include semiconductors,dielectrics, polymers, self-assembled biofilm, membranes, metals, alloy,and ceramics.

[0071] The sealing material is preferably deposited to surmount theplurality of channels to render such channels at least substantiallyenclosed, the substantially enclosed channels having a trench width ofless than 150 nanometers and a trench depth of less than 200 nanometers.By “surmount” is meant that the sealing material is preferentiallydeposited towards the top of the channels compared to the bottom of thechannels, resulting in substantially enclosed channels, which isdescribed above and in FIG. 4.

[0072] In certain embodiments of the present invention, the sealingmaterial can be deposited using any of a variety of methods, includingchemical vapor deposition, spin coating, film lamination, andthermo-evaporation. Preferably, the sealing material is deposited usingelectron-beam evaporation or sputtering.

[0073] In certain embodiments of the present invention, sealing materialis deposited on substrate surfaces by a sputtering process at gaspressures typically less than about 20 mTorr, more typically less than10 mTorr, and even more typically less than 5 mTorr. Sputtering is aprocess of driving molecules off a source target surface (such as SiO₂)using energetic ionic bombardment. Atoms are knocked off from the sourcetarget and can be deposited on a variety of substrate surfaces, such aspatterned silicon wafers. While not being bound to a particular theory,it is believed that as the gas pressure is reduced, there are fewerparticles in the environment of the plasma sputtering chamber, whichresults in the depositing atoms to travel with fewer collisions beforereaching the substrate surface; hence, a more anisotropic and fasterdeposition. At higher gas pressure such as 30 mTorr, depositing atomscollide more frequently on their path to the substrate surface, hence amore divergent traveling angles and more isotropic and slowerdeposition. At lower gas pressure with more anisotropic and fastdeposition, more depositing atoms can reach the bottom and lower part ofthe sidewalls of the trenches, causing relatively faster deposition ofsealing material at the bottom and sidewalls comparing to the top of thetrenches, this subsequently leads to smaller channel (trench) dimensions

[0074] The aspect ratio of the trenches being sealed also affects thegeometry of the final sealed void space. The higher the depth to widthratio, the less sealing material will be deposited near the bottom ofthe trench. The lower the depth to width ratio, the smaller and narrowerthe channel dimensions.

[0075] In one embodiment of carrying out the method of the presentinvention, at least one reservoir is provided to be in fluidcommunication with at least one end of at least one of the channels.Channels can be fabricated on the substrate using nanoimprinting andinterconnecting structures of pillar arrays. Reservoirs can be definedusing photolithography and subsequently pattern transferred to thesubstrate using Reactive Ion etching (RIE), chemical etching or FIBmilling directly to create reservoirs in fluid communication with thechannels. Auxiliary structures, such as microchannels, for connectingthe reservoirs to the channels can also be provided using these methods.Typical depth of the reservoirs and auxiliary structures is typically atleast several hundreds of nanometers, preferably at least severalmicrometers deep.

[0076] In certain embodiments, it is desirable to provide an additionalsealing step. A suitable additional sealing step includes application ofa planar surface substrate to the top of the sealing material.Alternatively, reservoirs can be formed on the sealing planar substrate.Auxiliary fluid communicating structures larger than about a micron canalso be formed to connect to larger sample reservoir. A variety ofschemes to connect reservoirs to the channels can be envisioned: atleast 2 reservoirs can be provided in fluid communication with at least2 separate channels; or at least 10 reservoirs are provided in fluidcommunication with at least 10 separate channels; or at least 96reservoirs are provided in fluid communication with at least 96 separatechannels; or at least 500 reservoirs are provided in fluid communicationwith at least 500 separate channels; or at least 5000 reservoirs areprovided in fluid communication with at least 100 separate channels; orcombinations thereof.

[0077] In a preferred embodiment of the present invention, the method ofpreparing the nanochannel arrays is carried out using linear channelarray substrates having a periodicity of less than 200 nm formed bynanoimprint lithography. In this embodiment, the linear channels have atrench width less than 100 nanometers and a trench depth less than 100nanometers. In this embodiment, at least a portion of the sealingmaterial is deposited using sputter deposition to provide sealingmaterial adjacent to the channel wall material to narrow the trenchwidth.

[0078] Varying the sealing material deposition parameters is also usedto narrow the trench width of the channels. The deposition parameterscan be varied to provide trench widths of typically less than 100nanometers. As more material is deposited, trench widths can be narrowedto less than 75 nanometers, and even less than: 50 nanometers, 25nanometers, and 15 nanometers. Trench widths of about 10 nm can also beprovided by the methods of the present invention. Typically, theresulting trench widths after deposition will be greater than 2 nm, andmore typically greater than 5 nanometers. Trench depths of less than175, 150, 125, 100, 75, 50, and 25 nm can also be provided by themethods of the present invention. Trench depths of about 15 nm can alsobe provided. Typically, the trench depths will be at least 5 nm, andmore typically at least 10 nm.

[0079] In another embodiment of the present invention, the method mayalso include the step of providing at least one near field slit featureabove at least one channel. In this step, the sealing material istypically transparent, such as silicon dioxide, to permit spectroscopicdetection of fluorescently labeled macromolecules, such as DNA, insidethe channels. This permits the use of optical methods, such asnear-field optical imaging, to analyze macromolecules in the channels.Nanochannel arrays suitable for near-field optical analysis can bemodified to have nanoslits. As described above, the nanoslit above thechannel is thin to permit sufficient evanescent excitation of thefluorescently-labeled macromolecules.

[0080] In one embodiment of the present invention, nanochannel arrayscan be prepared having a sufficiently thin seal thickness suitable fornear-filed optical analysis of fluids in the channels beneath thesealing material. In one embodiment, channels having an opaque sealingmaterial thicker than 100 nm can be modified using a suitablefabrication method to provide a nanoslit in the opaque sealing material.

[0081] Suitable fabrication methods for removing material from smallareas include E-beam lithography and Focus Ion Beam milling. E-beamlithography involves the lithography of ebeam resists followed bydevelopment and reactive ion etching. Focus Ion Beam (FIB) milling ispreferably used, as it requires fewer steps than E-beam lithography. FIBuses a beam of energetic ions such as Gallium ions to sputter materialaway and is capable of resolution down to 20 nm and can etch down manymicrons in principle. FIB is preferred as it enables one to image themilled area immediately after FIB milling the nanoslit structure.

[0082] In one embodiment of the present invention, a portion of thesealing material can be deposited inside the channels to form at leastone of: an insulating layer, a conducting layer, a hydrophilic layer, ahydrophobic layer, or combinations thereof. In this embodiment, thelayer thickness is typically less than half of the trench width.

[0083] In one embodiment, the dimensions, geometry, composition, orcombinations thereof, of the sealing material adjacent to the walls 114can be modified and manipulated for corresponding samples being analyzedin the channels. In a particular embodiment, it is desirable to alterthe surface properties of the sealing material adjacent to the wall 114This is carried out by treating at least some of the channels with asurface-modifying agent to alter the surfaces interior to said channels.

[0084] In one embodiment, surface-modifying agents are deposited in thechannels to improve the transport of macromolecules into and through thechannels. Surface modifying agents are particularly useful where theinternal dimensions (trench depth, trench height, or both) are less thanabout 50 nm. Surface-modifying agents can also reduce or increasehydrophobicity of the surfaces interior to said channels. Nanochannearrays made according to the present invention can be contacted withsolutions containing surface-modifying agents, such as by submerging thenanochannel array into such solutions. Suitable surface-modifying agentsinclude polyethyleneglycol (PEG), surfactants, Bovin Serum Albumin (BSA)protein solution, surface non-specific binding saturation, andanti-protein sticking agents. Application of a pressure differential,such as vacuum, can be used to assist the treatment of the channels.Application of vacuum is also useful for degassing any fluids inside thechannels.

[0085] In certain embodiments of the present invention, thesurface-modifying agent counteracts the electroosmosis effects insidethe channels. While not being bound to a particular theory, theelectroosmosis effect is usually due to ionized acidic groupsimmobilized to the matrix (e.g., attached to the wall) inducingpositively charged counter ions in the buffer that migrate towards thenegative electrodes, causing a bulk flow of liquid that migrates in thedirection opposite to that of the negatively charged DNA. Accordingly,reducing electroosmosis effects helps charged macromolecules to betransported into and along the channels.

[0086] In one embodiment of the present invention, the channels can beat least substantially enclosed on the surface of the substrate andsubstantially open on the edges of the substrate. As described herein,the channels are at least substantially enclosed by controlling thedeposition of the sealing material. In one embodiment, the channels aresubstantially open at the edges, which are readily provided by cleavingor cutting the substrate to reveal the interior portion of the channels.

[0087] In one embodiment, the deposition of the sealing materialcompletely encloses the plurality of channels. In this embodiment, thesealing layer is at least as thick as the atoms of the sealing material.Typically, the sealing material surmounting the plurality of channels isless than 500 nanometers thick. In certain embodiments, the sealingmaterial surmounting the plurality of channels can be less than: 100 nm,50 nm, 25 nm, 10 nm, and 5 nm thick. Typically the sealing materialsurmounting the plurality of channels is at least 1 nanometer thick, andmore typically at least 2 nm thick. In certain embodiments of thepresent invention, a step of removing a portion of the sealing materialis used to reduce the thickness of the sealing material above at leastone channel. Sealing material can be removed by a variety of etching andebeam deposition methods as further described herein.

[0088] In another aspect of the present invention, there is provided ananofluidic chip that includes a nanochannel array of the presentinvention. Referring to FIG. 5, the nanofluidic chip 200 has ananochannel array 100, a substrate 146, and reservoirs 144 for samplesand waste (or sample collection). Further provided in FIG. 5 areauxiliary sample ports 140 and auxiliary waste ports for handling fluidsample. The reservoirs are in fluid communication with at least one ofthe channels, so that the sample reservoirs are capable of releasing afluid into the channels, and the waste reservoirs are capable ofreceiving a fluid from the channels. Typically the fluids containmacromolecules for analysis.

[0089] In certain embodiments of the present invention, the nanofluidicchip contains at least one sample reservoir is formed in the surface ofthe substrate. Steps to form reservoirs in nanochannel array substratesare provided above. In this embodiment, at least one waste reservoir influid communication with at least one of the channels. Typically, thenanofluidic chip contains at least 1 sample reservoir. A variety ofother embodiments include at least 96 reservoirs, and even at least 1000reservoirs in the nanofluidic chip.

[0090] For use in macromolecular analysis, it is preferred that thenanofluidic chip provides at least a portion of the nanochannel arraycapable of being imaged with a two-dimensional detector. Imaging of thearray is provided by presenting the sealing material face of thenanochannel array to suitable apparatus for the collection of emittedsignals, such as optical elements for the collection of light from thenanochannel array. In this embodiment, the nanofluidic chip is capableof transporting a plurality of elongated macromolecules from a samplereservoir and across the channels.

[0091] In certain embodiments of the present invention, the nanofluidicchip contains an apparatus for transporting macromolecules from thesample reservoirs, through the channels, and into the waste reservoirs.A suitable apparatus includes at least one pair of electrodes capable ofapplying an electric field across at least some of the channels in atleast one direction. Electrode metal contacts can be integrated usingstandard integrated circuit fabrication technology to be in contact withat least one sample and at least one collection/waste reservoir toestablish directional electric field. Alternating current (AC), directcurrent (DC), or both types of fields can be applied. The electrodes canbe made of almost any metal, and are typically thin Al/Au metal layersdeposited on defined line paths. Typically at least one end of oneelectrode is in contact with buffer solution in the reservoir.

[0092] In certain embodiments of the present invention, the nanofluidicchip contains at least two pair of electrodes, each providing anelectric field in different directions. In this embodiment, adjacentclusters of channels connect individual isolated reservoir. With atleast two sets of independent electrodes, field contacts can be used toindependently modulate the direction and amplitudes of the electricfields to move macromolecules at desired speed or directions.

[0093] In another aspect of the present invention, there is provided asystem (FIG. 10, 300) that is suitable for carrying out macromolecularanalysis. In the present invention, the system includes a nanofluidicchip as described herein, and an apparatus for detecting at least onesignal transmitted from one or more fluids in the nanochannel array ofthe nanofluidic chip.

[0094] In various embodiments of the present invention, the systemfurther includes at least one of the following: a transporting apparatusto transport a fluid through at least one of the channels; a sampleloading apparatus for loading at least one fluid to the samplereservoirs in the nanofluidic chip; and a data processor. The variouscomponents of the system 300 are connected together, and the generalprinciples of operation are illustrated in FIG. 10.

[0095] The nanofluidic chip 200 used in the system is typicallydisposable, individually packaged, and having a sample loading capacityof 1-50,000 individual fluid samples. The nanofluidic chip typically hasat least one interconnecting sample delivery microchannel to providefluid samples into the channels, as well as sample loading openings anda reservoir, or sample loading openings and plates connected with asealing mechanism, such as an O-ring. Metal contacts for connecting theelectrodes 202 and an electric potential generator 216 are also providedin the nanofluidic chips. Suitable metal contacts can be externalcontact patches that can be connected to an externalscanning/imaging/electric-field tuner.

[0096] The nanofluidic chip is preferably encased in a suitable housing,such as plastic, to provide a convenient and commercially-readycartridge or cassette. Typically the nanofluidic cartridges will havesuitable features on or in the housing for inserting, guiding, andaligning the sample loading device with the reservoirs. Insertion slots,tracks, or both can be provided in the plastic case.

[0097] Macromolecular fluid samples that can be analyzed by the systemincludes fluids from a mammal (e.g., DNA, cells, blood, biopsy tissues),synthetic macromolecules such as polymers, and materials found in nature(e.g., materials derived from plants, animals, and other life forms).Such fluid samples can be managed, loaded, and injected using automatedor manual sample loading apparatus of the present invention.

[0098] In one embodiment of the present invention, the system includesan apparatus to excite the macromolecules inside the channels and detectand collect the resulting signals. A suitable apparatus is illustratedin FIG. 10: a laser beam 204 is focused using a focusing lens 206 to aspot on the nanochannel array 100. The generated light signal from themacromolecules inside the channels (not shown) is collected by acollection lens 208, and reflected off a dichroic mirror 218 into anoptical path 220, which is fed into a CCD (charge coupled device)camera. Various optical components and devices can also be used in thesystem to detect optical signals, such as digital cameras, PMTs(photomultiplier tubes), and APDs (Avalanche photodiodes.

[0099] In another embodiment of the present invention, the systemincludes a data processor. The data processor can be used to process thesignals from the CCD to project the digital image of the nanochannelarray on a display 212. The data processor can also analyze the digitalimage to provide characterization information, such as macromolecularsize statistics, histograms, karyotypes, mapping, diagnosticsinformation and display the information in suitable form for datareadout 214.

[0100] In another aspect of the present invention, there is provided amethod of analyzing at least one macromolecule. In this invention, theanalysis includes the steps of providing a nanofluidic chip according tothe present invention, providing the at least one sample reservoir withat least one fluid, said fluid comprising at least one macromolecule;transporting the at least one macromolecule into the at least onechannel to elongate said at least one macromolecule; detecting at leastone signal transmitted from the at least one elongated macromolecule;and correlating the detected signal to at least one property of the atleast one macromolecule.

[0101] In one embodiment of the present invention, the method ofanalyzing a macromolecule includes wetting the channels using capillaryaction with a buffer solution or a buffer solution containingmacromolecules. Macomolecules such as polymers and DNA can introducedinto nanochannel arrays by electric field.

[0102] Various macromolecules can be analyzed using the present method.For analyzing DNA typical process conditions include providing dilutesolutions of DNA which are stained at a ratio of 4:1 to 10:1 basepair/dye with a suitable dye. Suitable dye stains include TOTO-1,BOBO-1, BOBO-3 (Molecular Probes, Eugene, Oreg.). Solutions of stainedDNA can be further diluted and treated with an antioxidant and ananti-sticking agent.

[0103] In one embodiment of the present invention, the method ofanalyzing a macromolecule includes the sizing of one DNA. One DNAmacromolecule can be extracted from a single cell or spore, such asanthrax, and suitably transported (e.g., in a polymerized gel) to avoidbreakage.

[0104] Macromolecular fluid samples can be loaded through reservoirs inthe nanofluidics chip and transported via interconnecting microchannels.The macromolecules are partially elongated before one end of themacromolecule enters the channels; they are substantially fullyelongated when completely inside the channels. The fluorescent signalscan be excited by the appropriate excitation sources and emissionsignals can be collected via imaging camera or detectors, in a linearscanning mode or CCD image integration. The signals collected can beanalyzed by data processing software and user-defined major parameters(intensity/photons, major axis, minor axis, background signal)can berecorded and measured.

[0105] The length of a single DNA can be detected/reported and intensityprofile can be plotted. In various embodiments of the present invention,the method of analyzing a macromolecule includes correlating thedetected signal to at least one of the following properties: length,conformation, and chemical composition. Various macromolecules that canbe analyzed this way include, biopolymers such as a protein, apolypeptide, and a nucleic acid such as RNA or DNA For DNA nucleicacids, the detected signals can be correlated to the base pair sequenceof said DNA.

[0106] The typical concentration of the macromolecules in the fluid willbe one macromolecule, or about at least attogram per ml, more typicallyat least one femtogram per ml, more typically at least one picogram perml, and even more typically at least one nanogram per ml. Concentrationswill typically be less than 5 micrograms per milliliter and moretypically less than 0.5 micrograms per milliliter.

[0107] In one embodiment of the present invention, the method ofanalyzing a macromolecule measures the length of macromolecules havingan elongated length of greater than 150 nanometers, and typicallygreater than 500 nanometers, 1 micron, 10 microns, 100 microns, 1 mm, 1cm, and 10 cm long.

[0108] DNA having greater than 10 base pairs can also be analyzed usingthe present methods. Typically, the number of base pairs measured can begreater than 100 base pairs, greater than 1,000 base pairs, greater than10,000 base pairs, greater than 20,000 base pairs, greater than 40,000base pairs, and greater than 80,000 base pairs. DNA having more than 1million, 10 million, and even 100 million basepairs can be analyzed withthe present methods.

[0109] In one embodiment of the present invention, the methods can beused to analyze one or more of the following: restriction fragmentlength polymorphism, a chromosome, and single nucleotide polymorphism.

[0110] The following abbreviations are used herein: “nm” is nanometer,“mTorr” is milli Torr.

General Procedures

[0111] After NIL and etching, non-uniform deposition of sealing materialwas provided by e-beam evaporation with a tilted sample wafer at variousangles or sputter deposition using a large source target. This step wasused to both reduce the trench width and seal the channels.

[0112] Generally, 100-340 nm of SiO₂ was deposited onto the patternedsubstrate. Effective sealing was achieved with various depositionconditions that were tested. At gas pressure of 30 mTorr, RF power of900 W, and DC bias of 1400 V, a deposition rate of ˜9 nm/min wasachieved. At lower pressure of 5 mTorr, the deposition rate wasincreased to an estimated 17 nm/min. Sealing material was deposited onthe patterned substrate by sputtering at 5 mTorr.

EXAMPLES

[0113] In the following examples, nanochannel arrays were prepared usinga process to deposit SiO₂ sealing material on patterned substrates bysputtering. Channel openings were prepared by cleaving the substrate andimaged by Scanning Electronic Microscope (SEM). Results are as followsand shows that the trench widths are narrowed by the deposition of thesealing material using sputtering:

Example 1

[0114] A 100 mm silicon substrate was provided having a plurality ofparallel linear channels that had an 85 nm trench width and a 200 nmtrench height (FIG. 7a). This substrate was sputtered at a gas pressureof 5 mTorr according to the general procedures given above. Aftersputtering, the channels had a 52 nm trench width, a 186 nm trenchheight, and a seal thickness of 200 nm (FIG. 7b). Apparently, the trenchheight increased slightly as a result of the sealing process forming acone-shaped seal above the channel.

Example 2

[0115] A patterned substrate having a 65 nm trench width and about 100nm trench height prior to sputtering formed a nanochannel array having a17 nm trench width, a 68 nm trench height, and a channel seal thicknessof about 350 nm. Sputtering gas pressure was 5 mTorr.

Example 3

[0116] A patterned substrate having a 50 nm trench width and about 80 nmtrench depth prior to sputtering formed a nanochannel array having a 10nm trench width, 51 nm trench height, and a channel seal thickness of350 nm. Sputtering gas pressure was 5 mTorr.

Example 4

[0117] A substrate containing a two-dimensional array of pillars wasmade using a two-step NIL process with the channel mold rotated 90°between the imprinting steps (FIG. 6a). The pillar array structure issubsequently completely sealed with silicon dioxide using a 29 minutedeposition time. The seal thickness was about 500 nm. A profile view ofthe channel is depicted in FIG. 6b and a top view of the completelysealed nanochannel array is depicted in FIG. 6d.

Example 5

[0118] Example 4 was repeated except the sputter deposition time was 17minutes to provide a nanochannel array that is not completely sealed. Atop view scanning electron micrograph of this nanochannel array isprovided in FIG. 6d. The seal thickness was 300 nm.

Example 6

[0119] A nanoslit is provided in a channel prepared with a silicondioxide sealing material for carrying out near-field analysis having aseal thickness greater than about 100 nm is modified by using FIB tocreate a nanoslit having a thicknessless than 100 nm. FIG. 8 shows aschematic of how the deposited sealing material on a nanochannel arrayis first milled away using FIB from the sealing material situated abovethe sealed channels. Subsequently, aluminum is deposited to create anopaque layer to provide optical contrast at the slit.

Example 7

[0120] This example shows how a nanochannel array can be prepared from asubstrate having a plurality channels larger than 150 nm wide by 150 nmdeep. A substrate is prepared by photolithography techniques to providea plurality of channels with width of greater than 1.5 micron usingconventional optical lithography techniques: Contact aligner such asKarl Suss MA-6 to provide a pattern resolution at low micron level;Industrial projection stepper. The angle of the incident depositing beamof sealing material is varied to reduce the trench width and height toless than 150 nm and 150 nm, respectively, and to substantially seal byproviding shallow tangential deposition angles.

Example 8

[0121] This example provides a nanochannel array using an e-beamtechnique. A substrate is provided as in Example 1. Silicon dioxide isdeposited by an e-beam (thermo) evaporator (Temescal BJD-1800) onto thesubstrate. The substrate is placed at various angles incident to thedepositing beam from the silicon dioxide source target; the depositionrate is set to about 3 nm/minute and 150 nm of sealing material isdeposited in about 50 minutes.

Example 9

[0122] In this example, a nanochannel array is contacted with asurface-modifying agent. A nanochannel array made according to Example 1is submerged in a surface-modifying agents solutions containingpolyethylene glycol inside a vacuum chamber overnight to facilitatewetting and treatment of the channels and degas the air bubbles thatmight be trapped inside the channels.

Example 10

[0123] This example shows the preparation of a nanochannel array havinga metal sealing material. An e-beam (thermo) evaporator (TemescalBJD-1800) was used to deposit Chromium (Cr) onto a nanochannel arraychip (trench width 80 nm, trench depth 80 nm, SiO2/Si substrate). Thesubstrate was placed at various angles to the incident depositing beamfrom the source target, the deposition rate was set at 2.0-3.6nm/minute. The resulting trench width was 20 nm, trench depth less than80 nm, and the channels were substantially closed.

Example 11

[0124] This example shows the process of adding an optically opaquelayer to a nanochannel array. A nanochannel array made according toExample 3 is placed perpendicular to the incident depositing beam, toprovide an opaque layer less than 50 nm thick. An aluminum source targetis selected for depositing on top of the SiO2 sealing material above thesealed channels. The deposition rate was set at 2.0-3.6 nm/minute.

Example 12

[0125] This example describes the steps needed to provide a near-fieldslit in a nanochannel array. FIB was used to mill narrow slits less than50 nm in width in the direction perpendicular to the long axis of thenanochannel array of Example 11. The depth of the FIB milling wascontrolled to expose the underlying thin SiO2 sealing material above thenanochannel array.

Example 13

[0126] This example describes how to provide a sample reservoir with ananochannel array to provide a nanofluidic chip. A nanochannel arrayhaving 100 nm wide, 100 nm deep channels was made according to generalprocedures of Example 1. The nanochannel array was spin-coated with aphotoresist and imaged with a photomask to provide regions on oppositeends of the nanochannel array. The exposed areas were etched usingreactive ion etching to expose the channel ends and to provide amicron-deep reservoir about a millimeter wide on the opposite ends ofthe channels at the edge of the substrate.

Example 14

[0127] This example describes how to fill a nanofluidic chip with afluid containing DNA macromolecules to analyze the DNA. Acylindrical-shaped plastic sample-delivery tube of 2 mm diameter wasplaced in fluid communication with one of the reservoirs of thenanochannel array of Example 13. The delivery tube is connected to anexternal sample delivery/collection device, which is in turn connectedto a pressure /vacuum generating apparatus. The channels are wettedusing capillary action with a buffer solution. A buffer solutioncontaining stained lambda phage macromolecules (lambda DNA) wereintroduced into the nanochannel array by electric field (at 1-50 V/cm);the solution concentration was 5 microgram/mL and the lambda DNA wasstained at a ratio of 10:1 base pair/dye with the dye TOTO-1 (MolecularProbes, Eugene, Oreg.). This solution of stained DNA was diluted to0.1-0.5 microgram/mL into 0.5×TBE (tris-boroacetate buffer at pH 7.0)containing 0.1M of an anti-oxidant and 0.1% of a linear polyacrylamideused as an anti-sticking agent.

Example 14

[0128] A nanofluidic chip made according to Example 12, having channeldimensions of 100 nm×100 nm was filled using capillary action with abuffer solution containing stained genomic DNA to draw the DNAmacromolecules into the channels with an electric field. Bacteria phageDNA molecules Lambda (48.5 kb) and T4 (168.9 kb) were stained with thedye TOTO-1 and BOBO-3 respectively. This solution of stained DNA wasdiluted to 0.5 μg/mL into 0.5×TBE containing 0.1M dithiothreatol as ananti-oxidant and 0.1% of a linear acrylamide used as an antistickingagent). A Nikon Eclipse TE-300 inverted microscope with a 60× (N.A.1.4)oil immersion objective was used with an Ar:K laser (Coherent Lasers) asan excitation source at 488 nm and 570 nm. A Roper Scientific Pentamaxintensified cooled CCD camera with a 512×512 pixel array and 16 bitsdigital output was used to image the molecules. Digital image wasanalyzed using a data processor by NIH Image software. FIG. 9b shows anintegrated image of the stretched Lambda and T4 phage genomes side byside in the channels. The inset of 9 b shows the near perfect linear fitof the directly measured length obtained from the digital image plottedagainst their genome size, (R2 is 0.99996). FIG. 9c shows an array offluorescently-labeled genomic DNA molecules aligned and stretched in thechannels with the size ranging from 10 kb to 194 kb. This shows thatmillions of center-meter long parallel channels could be fabricated overthe whole wafer. Accordingly, the entire length of genomic DNA moleculescan be stretched and analyzed.

Example 15

[0129] Example 14 is repeated, but with a 96 multiple reservoir system.A nanofluidic chip made according to Example 12 is modified with aphotomask to provide 96 sample reservoirs, each reservoir connected to1000 channels along one edge of the 100 mm substrate. 96 different DNAsamples are delivered and injected using capillary fibers connected tothe sample reservoirs. 96 collection reservoirs are connected to thecorresponding ends of the channels to collect the DNA samples.

Example 16

[0130] This example describes a system used for carrying out analysis ofmacromolecules. The system contains an automated 96-capillaryauto-injection sample loader to deliver 96 macromolecular fluid samplesinto the delivery ports of a nanofluidic cartridge. The nanofluidiccartridge is a nanofluidic chip encased by a plastic polycarbonatehousing, having delivery ports and collection ports for connection tomicrocapillary tubing, and embedded metal contacts for connection toelectrodes on the nanofluidic chip. The cartridge can be inserted in acartridge holder, which is integrated with an a laser excitation sourceand suitable optical components to provide the excitation of andcollection of optical signals emanating from sample fluids within thenanochannel arrays of the nanofluidic chip. The signaldetection/collection apparatus is a cooled CCD digital camera. Signalsfrom the digital camera are analyzed by a data processor using NIH imageanalysis software, and displayed on a monitor.

Example 17

[0131] This example describes how to use the system of Example 16 tosize one DNA macromolecule. A single Anthrax spore is lysed to extractits entire genomic contents (DNA) with 10 microliters of a buffersolution and stained with fluorescent dyes. The sample loader isinserted into the delivery ports of the cartridge and injects the DNAcontaining fluid. The electrodes are activated and the DNAmacromolecules are transported into the nanochannel array, where theybecome elongated. The fluorescent stains on the DNA are excited by theexcitation source, and their emission signals are collected using theCCD camera. The signals collected, analyzed and recorded for intensityand position by the data processor. The length of a single DNA isdetected and intensity profile is plotted.

[0132] In another aspect of the present invention, there is provided144. A cartridge comprising at least one nanofluidic chip, saidcartridge capable of being inserted and removed from a system forcarrying out macromolecular analysis, said at least one nanofluidic chipcomprising at least one nanonanochannel array, said nanonanochannelarray comprising

[0133] a surface having a plurality of channels in the material of thesurface, said channels having a trench width of less than about 150nanometers and a trench depth of less than 200 nanometers;

[0134] at least some of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed.

We claim:
 1. A nanochannel array comprising a surface having a pluralityof channels in the material of the surface, said channels having atrench width of less than about 150 nanometers and a trench depth ofless than 200 nanometers; at least some of the channels being surmountedby sealing material to render such channels at least substantiallyenclosed.
 2. The nanochannel array of claim 1 wherein at least one endof at least one of the channels is in fluid communication with at leastone reservoir.
 3. The nanochannel array of claim 1 wherein both ends ofat least some of the channels are in fluid communication withreservoirs.
 4. The nanochannel array of claim 1 wherein the plurality ofchannels is in fluid communication with at least one sample reservoircommon to at least some of the channels.
 5. The nanochannel array ofclaim 4 wherein the plurality of channels is in fluid communication withat least one waste reservoir.
 6. The nanochannel array of claim 1wherein the channels are substantially parallel.
 7. The nanochannelarray of claim 1 wherein the ends of the channels are capable ofadmitting a macromolecule in a fluid.
 8. The nanochannel array of claim1 wherein the channels are capable of transporting a macromoleculeacross their length.
 9. The nanochannel array of claim 1 wherein thechannels are capable of transporting at least one macromolecule acrossthe length of said channels, said macromolecule being in an elongatedform,
 10. The nanochannel array of claim 1 wherein the channels arecapable of transporting at least one biopolymer across the length ofsaid channels.
 11. The nanochannel array of claim 10 wherein thechannels are capable of transporting at least one unfolded biopolymeracross the length of said channels.
 12. The nanochannel array of claim10 wherein the biopolymer is a nucleic acid.
 13. The nanochannel arrayof claim 10 wherein the biopolymer is an unfolded nucleic acid.
 14. Thenanochannel array of claim 1 wherein the trench width is less than 100nanometers.
 15. The nanochannel array of claim 1 wherein the trenchwidth is less than 75 nanometers.
 16. The nanochannel array of claim 1wherein the trench width is less than 50 nanometers.
 17. The nanochannelarray of claim 1 wherein the trench width is less than 25 nanometers.18. The nanochannel array of claim 1 wherein the trench width is lessthan 15 nanometers.
 19. The nanochannel array of claim 1 wherein thetrench width is about 10 nanometers.
 20. The nanochannel array of claim1 wherein the trench width is greater than 5 nanometers.
 21. Thenanochannel array of claim 1 wherein the trench width is greater than 2nanometers.
 22. The nanochannel array of claim 1 wherein the trenchdepth is less than 175 nanometers.
 23. The nanochannel array of claim 1wherein the trench depth is less than 125 nanometers.
 24. Thenanochannel array of claim 1 wherein the trench depth is less than 75nanometers.
 25. The nanochannel array of claim 1 wherein the trenchdepth is less than 50 nanometers.
 26. The nanochannel array of claim 1wherein the trench depth is less than 25 nanometers.
 27. The nanochannelarray of claim 1 wherein the trench depth is about 15 nanometers. 28.The nanochannel array of claim 1 wherein the trench depth is greaterthan 5 nanometers.
 29. The nanochannel array of claim 1 wherein thetrench depth is greater than 2 nanometers.
 30. The nanochannel array ofclaim 1 wherein the at least a portion of the channels are pillarstructures.
 31. The nanochannel array of claim 1 wherein the channelperiod is less than about 200 nm.
 32. The nanochannel array of claim 1wherein the material of the surface is a film adjacently supported by asecond substrate.
 33. The nanochannel array of claim 1 wherein thesubstrate material is a conducting material.
 34. The nanochannel arrayof claim 1 wherein the substrate material is a semi-conducting material.35. The nanochannel array of claim 1 wherein the substrate material is anon-conducting material.
 36. The nanochannel array of claim 1 furthercomprising at least one optically opaque layer adjacent to the sealingmaterial.
 37. The nanochannel array of claim 1 further comprising atleast one near field slit above at least one channel.
 38. Thenanochannel array of claim 1 further comprising sealing materialadjacent to a channel bottom.
 39. The nanochannel array of claim 1further comprising sealing material adjacent to a channel wall.
 40. Thenanochannel array of claim 1 wherein the dimensions of the sealingmaterial are such that Δw′ is greater than or equal to Δw″.
 41. Thenanochannel array of claim 1 wherein the length of the channels isgreater than 1 millimeter.
 42. The nanochannel array of claim 1 whereinthe length of the channels is greater than 1 centimeter.
 43. Thenanochannel array of claim 1 wherein the length of the channels isgreater than 5 centimeters.
 44. The nanochannel array of claim 1 whereinthe length of the channels is greater than 15 centimeters.
 45. Thenanochannel array of claim 1 wherein the length of the channels isgreater than 25 centimeters.
 46. The nanochannel array of claim 1wherein the substrate is a flexible film material.
 47. A method ofpreparing a nanochannel array, comprising the steps of: providing asubstrate having a surface; forming a plurality of channels in thematerial of the surface; and depositing a sealing material on theplurality of channels to surmount the plurality of channels to rendersuch channels at least substantially closed, the substantially closedchannels having a trench width of less than about 150 nanometers and atrench depth of less than 200 nanometers.
 48. The method of claim 47further comprising the step of providing at least one reservoir in fluidcommunication with at least one end of at least one of the channels. 49.The method of claim 48 wherein at least 2 reservoirs are provided influid communication with at least 2 separate channels.
 50. The method ofclaim 48 wherein at least 10 reservoirs are provided in fluidcommunication with at least 10 separate channels.
 51. The method ofclaim 48 wherein at least 96 reservoirs are provided in fluidcommunication with at least 96 separate channels.
 52. The method ofclaim 48 wherein at least 500 reservoirs are provided in fluidcommunication with at least 500 separate channels.
 53. The method ofclaim 47 wherein the plurality of channels is formed by: nanoimprintlithography, spin coating, electron beam lithography, focused ion beammilling, photolithography, reactive ion-etching, wet-etching,plasma-enhanced chemical vapor deposition, electron beam evaporation,sputter deposition, and combinations thereof.
 54. The method of claim 47wherein the channels are linear channels formed by nanoimprintlithography, said linear channels having a trench width less than 100nanometers and a trench depth less than 175 nanometers, wherein at leasta portion of the sealing material is deposited using sputter depositionto provide sealing material adjacent to the channel wall material tonarrow the trench width.
 55. The method of claim 47 wherein the trenchwidth is less than 100 nanometers.
 56. The method of claim 47 whereinthe trench width is less than 75 nanometers.
 57. The method of claim 47wherein the trench width is less than 50 nanometers.
 58. The method ofclaim 47 wherein the trench width is less than 25 nanometers.
 59. Themethod of claim 47 wherein the trench width is less than 15 nanometers.60. The method of claim 47 wherein the trench width is about 10nanometers.
 61. The method of claim 47 wherein the trench width isgreater than 5 nanometers.
 62. The method of claim 47 wherein the trenchwidth is greater than 2 nanometers.
 63. The method of claim 47 whereinthe trench depth is less than 175 nanometers.
 64. The method of claim 47wherein the trench depth is less than 125 nanometers.
 65. The method ofclaim 47 wherein the trench depth is less than 100 nanometers.
 66. Themethod of claim 47 wherein the trench depth is less than 75 nanometers.67. The method of claim 47 wherein the trench depth is less than 50nanometers.
 68. The method of claim 47 wherein the trench depth is about15 nanometers.
 69. The method of claim 47 wherein the trench depth isgreater than 5 nanometers.
 70. The method of claim 47 wherein the trenchdepth is greater than 2 nanometers.
 71. The method of claim 47 whereinthe sealing material is deposited using electron beam evaporation orsputter deposition.
 72. The method of claim 47 wherein the plurality ofchannels are formed substantially parallel.
 73. The method of claim 47wherein the plurality of channels are pillar structures.
 74. The methodof claim 47 wherein the channels in the substrate material have aperiodicity of less than about 200 nm.
 75. The method of claim 47wherein the material of the surface is a film adjacently supported by asecond substrate.
 76. The method of claim 74 wherein the substratematerial is conductive.
 77. The method of claim 74 wherein the substratematerial is semiconductive.
 78. The method of claim 74 wherein thesubstrate material is non-conductive.
 79. The method of claim 47 furthercomprising the step of depositing at least one optically opaque layeradjacent to the sealing material.
 80. The method of claim 79 wherein thestep of depositing at least one optically opaque layer occurs prior todepositing the sealing material.
 81. The method of claim 79 wherein thestep of depositing at least one optically opaque layer occurs subsequentto depositing the sealing material.
 82. The method of claim 47 furthercomprising the step of providing at least one near field slit featureabove at least one channel.
 83. The method of claim 47 wherein a portionof the sealing material is deposited within the channels.
 84. The methodof claim 83 wherein a portion of the sealing material is deposited inthe bottom of at least one channel.
 85. The method of claim 83 wherein aportion of the sealing material is deposited on at least one wall of atleast one channel.
 86. The method of claim 47 wherein the dimensions ofthe deposited sealing material are such that Δw′ is greater than orequal to Δw″.
 87. The method of claim 47 further comprising the step oftreating at least some of the channels with a surface-modifying agent toalter the surfaces interior to said channels.
 88. The method of claim 87wherein the surface-modifying agent reduces hydrophobicity of thesurfaces interior to said channels.
 89. The method of claim 87 whereinthe surface-modifying agent increases hydrophobicity of the surfacesinterior to said channels.
 90. The method of claim 87 wherein thesurface-modifying agent counteracts the electroosmosis effects insidethe channels.
 91. The method of claim 47 wherein the channels aresubstantially closed on the surface of the substrate and substantiallyopen on the edges of the substrate.
 92. The method of claim 47 whereinthe sealing material surmounting the plurality of channels is at leastthe thickness of the atoms of the sealing material.
 93. The method ofclaim 47 wherein the sealing material surmounting the plurality ofchannels is less than 500 nanometers thick.
 94. The method of claim 47wherein the sealing material surmounting the plurality of channels isless than 150 nanometers thick.
 95. The method of claim 47 wherein thesealing material surmounting the plurality of channels is less than 100nanometers thick.
 96. The method of claim 47 wherein the sealingmaterial surmounting the plurality of channels is less than 50nanometers thick.
 97. The method of claim 47 wherein the sealingmaterial surmounting the plurality of channels is less than 25nanometers thick.
 98. The method of claim 47 wherein the sealingmaterial surmounting the plurality of channels is less than 10nanometers thick.
 99. The method of claim 47 wherein the sealingmaterial surmounting the plurality of channels is less than 5 nanometersthick.
 100. The method of claim 47 wherein the sealing materialsurmounting the plurality of channels is at least 1 nanometer thick.101. The method of claim 47 wherein the sealing material surmounting theplurality of channels is at least 2 nanometers thick.
 102. The method ofclaim 47 further comprising the step of removing a portion of thesealing material to reduce the thickness of the sealing material aboveat least one channel.
 103. A nanofluidic chip, comprising: a)nanochannel array, comprising: a substrate having a surface; a pluralityof parallel channels in the material of the surface, said channelshaving a trench width of less than about 150 nanometers and a trenchdepth of less than 200 nanometers; at least some of the channels beingsurmounted by sealing material to render such channels at leastsubstantially enclosed; at least some of the channels are capable ofadmitting a fluid; b) at least one sample reservoir in fluidcommunication with at least one of the channels, said sample reservoircapable of releasing a fluid; and c) at least one waste reservoir influid communication with at least one of the channels, said wastereservoir capable of receiving a fluid.
 104. The nanofluidic chip ofclaim 103 wherein the at least one sample reservoir is formed in thesurface of the substrate.
 105. The nanofluidic chip of claim 103 furthercomprising at least one waste reservoir in fluid communication with atleast one of the channels.
 106. The nanofluidic chip of claim 103wherein the number of sample reservoirs is at least
 10. 107. Thenanofluidic chip of claim 103 wherein the number of sample reservoirs isat least
 96. 108. The nanofluidic chip of claim 103 wherein the numberof sample reservoirs is at least
 1000. 109. The nanofluidic chip ofclaim 103 wherein at least a portion of the nanochannel array capable ofbeing imaged with a two-dimensional detector.
 110. The nanofluidic chipof claim 103 capable of transporting a plurality of elongatedmacromolecules from a sample reservoir and across the channels.
 111. Thenanofluidic chip of claim 103 further comprising at least one pair ofelectrodes capable of applying an electric field across at least some ofthe channels in at least one direction.
 112. The nanofluidic chip ofclaim 111 wherein at least two pair of electrodes each provides anelectric field in different directions.
 113. A system, comprising: A) ananofluidic chip, comprising: a) nanochannel array, comprising: asubstrate having a surface; a plurality of parallel channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least one of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast one of the channels capable of admitting a fluid; and b) at leastone sample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid; and B) anapparatus for detecting at least one signal transmitted from the atleast one fluid in the nanochannel array.
 114. The system according toclaim 113, further comprising a transporting apparatus to transport afluid through at least one of the channels.
 115. The system according toclaim 113, further comprising a sample loading apparatus for loading atleast one fluid to the at least one sample reservoir,
 116. The systemaccording to claim 113, further comprising a data processor.
 117. Amethod of analyzing at least one macromolecule, comprising the steps of:providing a nanofluidic chip, comprising: a) nanochannel array,comprising: a surface having a plurality of parallel channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least one of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed; atleast one of the channels capable of admitting a fluid; b) at least onesample reservoir in fluid communication with at least one of thechannels, said sample reservoir capable of releasing a fluid containingat least one macromolecule; providing the at least one sample reservoirwith at least one fluid, said fluid comprising at least onemacromolecule; transporting the at least one macromolecule into the atleast one channel to elongate said at least one macromolecule; detectingat least one signal transmitted from the at least one elongatedmacromolecule; and correlating the detected signal to at least oneproperty of the at least one macromolecule.
 118. The method according toclaim 114 wherein the detected signal is correlated to at least one ofthe following properties: length, conformation, and chemicalcomposition.
 119. The method according to claim 114 wherein themacromolecule is a synthetic polymer or biopolymer.
 120. The method ofclaim 116 wherein the biopolymer is at least one of: a protein, apolypeptide, and a nucleic acid.
 121. The method of claim 117 whereinthe nucleic acid is DNA and the detected signals are correlated to thebase pair sequence of said DNA.
 122. The method of claim 114 wherein aplurality of reservoirs provide a plurality of macromolecules into aplurality of channels for determining the lengths of the macromolecules.123. The method of claim 119 wherein more than one of the macromoleculesenters a single channel.
 124. The method of claim 119 wherein themacromolecules are biopolymers.
 125. The method of claim 121 wherein thebiopolymers are proteins, polypeptides, DNA or RNA.
 126. The method ofclaim 121 wherein the biopolymers are at least substantially unfolded inthe channels.
 127. The method of claim 114 wherein the concentration ofthe macromolecules in the fluid is at least one attogram per milliliter.128. The method of claim 114 wherein the concentration of themacromolecules in the fluid is at least one femtogram per milliliter.129. The method of claim 114 wherein the concentration of themacromolecules in the fluid is at least one picogram per milliliter.130. The method of claim 114 wherein the concentration of themacromolecules in the fluid is less than 5 micrograms per milliliter.131. The method of claim 114 wherein the concentration of themacromolecules in the fluid is less than 0.5 micrograms per milliliter.132. The method of claim 114 wherein the macromolecules have anelongated length in the channels of greater than 150 nanometers. 133.The method of claim 114 wherein the macromolecules have an elongatedlength in the channels of greater than 500 nanometers.
 134. The methodof claim 114 wherein the macromolecules have an elongated length in thechannels of greater than 1 micron.
 135. The method of claim 114 whereinthe macromolecules have an elongated length in the channels of greaterthan 10 microns.
 136. The method of claim 114 wherein the macromoleculesare DNA having greater than 10 base pairs.
 137. The method of claim 114wherein the macromolecules are DNA having greater than 100 base pairs.138. The method of claim 114 wherein the macromolecules are DNA havinggreater than 1,000 base pairs.
 139. The method of claim 114 wherein themacromolecules are DNA having greater than 10,000 base pairs.
 140. Themethod of claim 114 wherein the macromolecules are DNA having greaterthan 20,000 base pairs.
 141. The method of claim 114 wherein themacromolecules are DNA having greater than 40,000 base pairs.
 142. Themethod of claim 114 wherein the macromolecules are DNA having greaterthan 80,000 base pairs.
 143. The method of claim 114 wherein thenanochannel array has at least 96 sample simultaneously reservoirs foranalyzing at least 96 different macromolecular fluid samples.
 144. Themethod of claim 114 wherein the at least one macromolecule is arestriction fragment length polymorphism.
 145. The method of claim 114wherein the at least one macromolecule is a chromosome.
 146. The methodof claim 142 wherein the at least one chromosome is analyzed todetermine the presence of at least one single nucleotide polymorphism.147. A cartridge comprising at least one nanofluidic chip, saidcartridge capable of being inserted and removed from a system forcarrying out macromolecular analysis, said at least one nanofluidic chipcomprising at least one nanonanochannel array, said nanonanochannelarray comprising a surface having a plurality of channels in thematerial of the surface, said channels having a trench width of lessthan about 150 nanometers and a trench depth of less than 200nanometers; at least some of the channels being surmounted by sealingmaterial to render such channels at least substantially enclosed.